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The Latest Research Items

Protein Stability in Living Cells

The intracellular milieu differs from the dilute conditions in which most biophysical and biochemical studies are performed. This difference has led both experimentalists and theoreticians to tackle the challenging task of understanding how the intracellular environment affects the properties of biopolymers. Despite a growing number of in-cell studies, there is a lack of quantitative, residue-level information about equilibrium thermodynamic protein stability under nonperturbing conditions.

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William Monteith and Professor Gary Pielak, published in PNAS, report the use of NMR-detected hydrogen–deuterium exchange of quenched cell lysates to measure individual opening free energies of the 56-aa B1 domain of protein G (GB1) in living Escherichia coli cells without adding destabilizing cosolutes or heat. Comparisons to dilute solution data, pH 7.6 and 37 °C, show that opening free energies increase by as much as 1.14 ± 0.05 kcal/mol in cells. Importantly, this research also shows that homogeneous protein crowders destabilize GB1, highlighting the challenge of recreating the cellular interior. William and Gary discuss their findings in terms of hard-core excluded volume effects, charge–charge GB1-crowder interactions, and other factors. The quenched lysate method identifies the residues most important for folding GB1 in cells, and should prove useful for quantifying the stability of other globular proteins in cells to gain a more complete understanding of the effects of the intracellular environment on protein chemistry.

 

Switchable Micropatterned Surfaces

Researchers in the Ashby and Sheiko groups have fabricated textured surfaces capable of reversibly changing in response to a thermal stimulus. These surfaces are fabricated with PRINT© molds provided by the DeSimone Lab, and could find use in applications requiring modular surface wetting or roughness. Reversibly switching topography on micrometer length scales greatly expands the functionality of stimuli-responsive substrates.

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In an article published in ACS Applied Materials & Interfaces the groups report the first usage of reversible shape memory for the actuation of two-way transitions between microscopically patterned substrates, resulting in corresponding modulations of the wetting properties. Reversible switching of the surface topography is achieved through partial melting and recrystallization of a semi-crystalline polyester embossed with microscopic features. This behavior is monitored with atomic force microscopy, AFM, and contact angle measurements. The groups demonstrate that the magnitude of the contact angle variations depends on the embossment pattern.

 

Reducing Drug Toxicity with PRINT

The synthesis of prodrugs is a common approach to overcome drug delivery issues, including poor aqueous solubility or permeability, and to provide site-specific release. Nanotechnology can be a powerful tool to improve drug delivery, but does so by altering the biodistribution of the encapsulated small molecule. In a report published in NanoLetters, researchers in the DeSimone Group, in collaboration with a number of Centers, Institutes, and Departments here at UNC, combined the merits of both approaches to improve the pharmacokinetics and toxicity of the chemotherapeutic docetaxel by passively targeting an encapsulated docetaxel prodrug to solid tumors, where it could selectively release and convert to active docetaxel.

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The Group used PRINT technology, Particle Replication in Nonwetting Templates, to prepare nanoparticles to passively target solid tumors in an A549 subcutaneous xenograft model. An acid labile prodrug was delivered to minimize systemic free docetaxel concentrations and improve tolerability without compromising efficacy.

 

Electrocatalytic Carbon Dioxide Reduction

Accumulation of carbon dioxide in the atmosphere is considered a major contributor to climate change. Once captured, CO2 is a potentially useful feedstock if it can be converted into formate/formic acid, carbon monoxide, or more highly reduced hydrocarbon products. Electrochemical and photoelectrochemical CO2 reduction could become an integral part of an energy storage strategy with solar- or wind-generated electricity used to store energy in the chemical bonds of carbon-based fuels.

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The Meyer Group, in collaboration with the Department of Electrical and Computer Engineering at Duke University, published in JACS, reports on how Nitrogen-doped carbon nanotubes are selective and robust electrocatalysts for CO2 reduction to formate in aqueous media without the use of a metal catalyst. An overlayer of polyethylenimine (PEI) functions as a cocatalyst by significantly reducing catalytic overpotential and increasing current density and efficiency.

 

Trifluormethyl Pyrrolidines

Researchers in the Johnson Group, published in Organic Letters, describe the stereoselective synthesis of trisubstituted 2-trifluoromethyl pyrrolidines by asymmetric Michael addition/hydrogenative cyclization.

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The direct organocatalytic addition of 1,1,1-trifluoromethylketones to nitroolefins proceeds under mild reaction conditions and low catalyst loadings to provide Michael adducts in high yield with excellent diastereo- and enantioselectivity. Catalytic hydrogenation of the Michael adducts stereoselectively generates 2-trifluoromethylated pyrrolidines bearing three contiguous stereocenters. The group members also describe a stereospecific route to epimeric 2-trifluoromethyl pyrrolidines from a common intermediate.

 

Chemical Vapor Deposition

Researchers in the Ramsey Group, published in Analytical Chemistry, describe a chemical vapor deposition, CVD, method for the surface modification of glass microfluidic devices designed to perform electrophoretic separations of cationic species. The microfluidic channel surfaces were modified using aminopropyl silane reagents. Coating homogeneity was inferred by precise measurement of the separation efficiency and electroosmotic mobility for multiple microfluidic devices.

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Microfluidic devices with a 23 cm long, serpentine electrophoretic separation channel and integrated nanoelectrospray ionization emitter were CVD coated with (3-aminopropyl)di-isopropylethoxysilane, APDIPES, and used for capillary electrophoresis (CE)-electrospray ionization (ESI)-mass spectrometry (MS) of peptides and proteins. Peptide separations were fast and highly efficient, yielding theoretical plate counts over 600,000 and a peak capacity of 64 in less than 90 s. Intact protein separations using these devices yielded Gaussian peak profiles with separation efficiencies between 100,000 and 400,000 theoretical plates.

 

Small Sample Sorting

Primary patient samples are the gold standard for molecular investigations of tumor biology yet are difficult to acquire, heterogeneous in nature and variable in size. Patient-derived xenografts, PDXs, comprised of primary tumor tissue cultured in host organisms such as nude mice permit the propagation of human tumor samples in an in vivo environment and closely mimic the phenotype and gene expression profile of the primary tumor. Although PDX models reduce the cost and complexity of acquiring sample tissue and permit repeated sampling of the primary tumor, these samples are typically contaminated by immune, blood, and vascular tissues from the host organism while also being limited in size.

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For very small tissue samples, on the order of 103 cells, purification by fluorescence-activated cell sorting, FACS, is not feasible while magnetic activated cell sorting, MACS, of small samples results in very low purity, low yield, and poor viability. Researchers in the Allbritton Group have now developed a platform for imaging cytometry integrated with micropallet array technology to perform automated cell sorting on very small samples obtained from PDX models of pancreatic and colorectal cancer using antibody staining of EpCAM, CD326, as a selection criteria. Published in Cytometry Part A, the data collected demonstrate the ability to automate and efficiently separate samples with very low number of cells.

 

Dual Action Antibacterial Agents

Published in Bioconjugate Chemistry, researchers in the Schoenfisch Group describe the synthesis of nitric oxide, NO, releasing quaternary ammonium, QA, functionalized generation 1, G1, and generation 4, G4, poly(amidoamine), PAMAM, dendrimers. Dendrimers were modified with QA moieties of different alkyl chain lengths, such as methyl, butyl, octyl, dodecyl, via a ring-opening reaction. The resultant secondary amines were then modified with N-diazeniumdiolate NO donors to yield NO-releasing QA-modified PAMAM dendrimers capable of spontaneous NO release.

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The bactericidal efficacy of individual, non-NO-releasing, and dual action, NO-releasing, QA-modified PAMAM dendrimers was evaluated against Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa bacteria. Bactericidal activity was found to be dependent on dendrimer generation, QA alkyl chain length, and bacterial Gram class for both systems. Shorter alkyl chains, such as methylQA and butylQA, demonstrated increased bactericidal activity against P. aeruginosa versus S. aureus for both generations, with NO release markedly enhancing overall killing.

 

Organ-on-a-Chip

New advances enable long-term organotypic culture of colonic epithelial stem cells that develop into structures known as colonoids. Colonoids represent a primary tissue source acting as a potential starting material for development of an in vitro model of the colon. Key features of colonic crypt isolation and subsequent colonoid culture have not been systematically optimized compromising efficiency and reproducibility. Research from the Allbritton Group, published in the Journal of Biological Engineering, show how murine crypt isolation yield and quality can be optimized, and colonoid culture efficiency measured in microfabricated culture devices.

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Improved crypt isolation and 3-D colonoid culture, along with an understanding of colonic epithelial cell behavior in the presence of microfabrication substrates will support development of "organ-on-a-chip" approaches for studies using primary colonic epithelium.

 

Immobilization of Lambda Exonuclease

The process of immobilizing enzymes onto solid supports for bioreactions has some compelling advantages compared to their solution-based counterpart including the facile separation of enzyme from products, elimination of enzyme autodigestion, and increased enzyme stability and activity. Researchers in the Soper Group, published in Analytical Chemistry report the immobilization of λ-exonuclease onto poly(methylmethacrylate) (PMMA) micropillars populated within a microfluidic device for the on-chip digestion of double-stranded DNA.

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The group's results suggest that the efficiency for the catalysis of dsDNA digestion using λ-exonuclease, including its processivity and reaction rate, were higher when the enzyme was attached to a solid support compared to the free solution digestion. The results from this work will have important ramifications in new single-molecule DNA sequencing strategies that employ free mononucleotide identification.

 

Imaging Charge Separation in Nanowires

Silicon nanowires incorporating p-type/n-type (p n) junctions have been introduced as basic building blocks for future nanoscale electronic components. Controlling charge flow through these doped nanostructures is central to their function, yet our understanding of this process is inferred from measurements that average over entire structures or integrate over long times.

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Published in Nano Letters, researchers from the Cahoon and Papanikolas Groups describe how they used femtosecond pump-probe microscopy to directly image the dynamics of photogenerated charge carriers in silicon nanowires encoded with p-n junctions along the growth axis. Initially, motion is dictated by carrier-carrier interactions, resulting in diffusive spreading of the neutral electron-hole cloud. Charge separation occurs at longer times as the carrier distribution reaches the edges of the depletion region, leading to a persistent electron population in the n type region. Time-resolved visualization of the carrier dynamics yields clear, direct information on fundamental drift, diffusion, and recombination processes in these systems, providing a powerful tool for understanding and improving materials for nanotechnology.

 

Controlling Molecular Weight

Molecular weight, MW, its distribution and dispersity, PDI, of polymers are possibly the most important characteristics that distinguish polymers from small organic molecules. Conjugated polymers are no exception to this. For polymer solar cells, a high MW is usually desirable. For example, high MW polymers have good viscosity desirable for thin films coating. More importantly, it appears that a high MW is beneficial for a higher current of solar cells. However, a number of questions had still remained to be answered, such as: Is there any appropriate MW for conjugated polymers used for solar cells? If so, can we control the MW? What about PDI?

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Published in Advanced Materials, the You Group offers some insights towards these outstanding issues concerning MW. Taking their well-acclaimed conjugated polymer, PBnDT-FTAZ, as the model system, they created a set of polymers with precisely controlled MW by adjusting stoichiometric ratio of two monomers, following the classic Carothers question. In collaboration with the Ade Group at NCSU, the You Group carefully investigated this set of PBnDT-FTAZ with different MW and discovered that the MW significantly influences the morphology and structural order of PBnDT-FTAZ in its bulk heterojunction solar cells, with highest efficiency, over 7%, resulting with use of a MW of 40 kg/mol. Additionally, by recreating a 40 kg/mol polymer with a higher PDI of 3.2 than the pristine 40 kg/mol polymer, PDI of 2.2, they showed that the dispersity,PDI, though largely neglected in the past, might play a role in affecting the device performance of polymer solar cells.